Abstract Endometrioid-type endometrial carcinoma (EEC) accounts for approximately 75% of all endometrial carcinomas, the fourth most common cancer in women in the US. Many patients with early stage and low grade EEC will be cured by surgery alone but for women who present with higher grade, advanced stage EEC, more aggressive therapeutics are needed to control the disease. From TCGA data (and validated from our own patients) we have identified four transcriptome subtypes of EEC with distinct clinicopathologic characteristics and mutation spectra. Cluster II consists of younger, obese patients with low grade EEC yet diminished survival. Although the Cluster II tumors had the lowest overall mutation rate, CTNNB1 exon 3 mutations were very common. These mutations were associated with activation of the Wnt/?-catenin signaling pathway. We hypothesize that mutations in exon 3 of CTNNB1 reprogram the molecular landscape leading to clinically aggressive EEC. Understanding of these mutations will better inform specific strategies targeting the Wnt pathway including cyclin D and CDK4. The following specific aims are proposed to test this hypothesis. Specific Aim 1. Test the hypothesis that exon 3 mutations of CTNNB1 alter cellular epigenetic programs, thus suppressing the hormonal gene expression program and activating a mesenchymal/neuronal and immunosuppressive gene expression programs. 1.1. Functionally characterize the 7 hot spot mutants by establishing isogenic stable lines expressing each of the mutants. 1.2. Characterize the regulatory network of mutant CTNNB1 by transcriptome and miRNA profiling of the stable lines and identify the target genes, initially focusing on ESR1, PGR, N-cadherin, PDGFA, WNT5A, WNT5B, IL-10, and TGFB2. 1.3. Determine the possible driver effect of N-cadherin, WNT5A, and WNT5B by gain of function or loss of function via siRNA (individually or in combinations) and examining relevant in vitro cellular endpoints. Specific Aim 2. Establish preclinical and clinical models to test the hypothesis that activating mutations of CTNNB1 are important drivers of tumorigenesis. 2.1. Evaluate the effects of CTNNB1 mutation in vivo. 2.2. Utilize a 3D in vitro system to test alternative therapeutics targeting Wnt/?-catenin signaling. 2.3. Determine if CTNNB1 mutation promotes the formation of an immunosuppressive microenvironment in hysterectomy specimens. Specific Aim 3. Conduct a phase II, single arm therapeutic trial of ribociclib (Novartis CDK4/6 inhibitor), letrozole, and everolimus for advanced/recurrent EEC. We have previously shown that letrozole+everolimus is effective in a subset of these patients. Cyclin D1 is one of the highest induced proteins in CTNNB1-mutated endometrial carcinomas, and it interacts with CDK4/6 to promote cell cycle progression. We hypothesize that patients with carcinomas with CTNNB1 mutation will have higher expression of Cyclin D1 and therefore be more responsive to treatment with this combination. The trial will be enriched for patients with tumors with CTNNB1 mutations.